The goal of our studies is to generate robust and long-lasting tumor-specific T cell responses for durable tumor regression in patients with chemotherapy-resistant epithelial ovarian cancer (EOC). Although immunotherapy using immune checkpoint blockade, adoptive T cell therapy, or oncolytic viruses has generated remarkable results in several other tumor types, long-term tumor control has been infrequent in EOC patients. Studies by our group have identified key stumbling blocks underpinning the limited efficacy of immunotherapy in EOC, which include: (i) insufficient expansion of tumor-specific T cells, (ii) tumor-derived CXCL12 production that recruits Tregs and MDSC within tumors, (iii) expression of PD1 and LAG3 marking severely dysfunctional tumor infiltrating T lymphocytes (TILs), (iv) compensatory upregulation of LAG3 upon blockade of PD1 and vice-versa, and (v) disorganized and tortuous tumor vasculature lacking adequate flow dynamics to support trafficking of anti-tumor T cells. While combinatorial immunotherapy strategies have the potential to overcome these immune resistance mechanisms in the tumor microenvironment (TME), they are often associated with unacceptably high rates of toxicities in patients. Our proposal addresses these stumbling blocks using innovative, clinically-translatable strategies to reprogram the TME and to identify mechanisms that drive or hinder T cell trafficking into ovarian tumors. We previously demonstrated blockade of the CXCL12/CXCR4 axis in the ovarian TME by intraperitoneal delivery of an oncolytic vaccinia virus expressing a CXCR4 antagonist (OV-CXCR4-Fc) reduced intratumoral accumulation of immunosuppressive mediators and stimulated spontaneous antitumor immunity to endogenous tumor antigens. OV-CXCR4-Fc also improved T cell trafficking. We will build on the multifaceted antitumor activity of OV-CXCR4-Fc by further ?arming? with antagonists of PD1 and LAG3, the two co-inhibitory receptors we have shown mark severely dysfunctional TILs in EOC patients. In addition, we plan to uncover the molecular mechanisms by which OV-CXCR4-Fc enhances T cell migration and trafficking to overcome the ovarian tumor ?vascular checkpoint?. We hypothesize that in vivo tumor destruction by OV engineered to express the CXCR4 antagonist and PD1/LAG3 inhibitors (OV-CXCR4-Fc-PD1/LAG3) will limit dampening of the immune response in the TME and induce a molecular signature that promotes trafficking and homing of tumor-specific T cells. To test our hypotheses, we propose the following specific aims: Aim 1. Determine the therapeutic synergy of arming OV-CXCR4-Fc with PD1/LAG3 inhibitors. Aim 2. Determine the mechanism by which OV-CXCR4-Fc promotes T cell trafficking in murine and human ovarian tumors. Aim 3. To manufacture and certify a Master Viral Bank (MVB) and Final Product (FP) of OV-CXCR4-Fc for IND qualification.